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Mechanism-Based Inactivation of Humam Neutrophil Elastase

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Mechanism-Based Inactivation of Humam Neutrophil Elastase MECHANISM-BASED INACTIVATION OF HUMAM NEUTROPHIL ELASTASE A Thesis by Yi Li B. S., Jilin University, 2005 Submitted to the Department of Chemistry and the faculty and Graduate School of Wichita State University in partial fulfillment of the requirements for the degree of Master of Science December 2008 © Copyright 2008 by Yi Li, All Rights Reserved ii MECHANISM-BASED INACTIVATION OF HUMAM NEUTROPHIL ELASTASE I have examined the final copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirement for the degree of Master of Science with a major in Chemistry _________________________________ William C. Groutas, Committee Chair We have read this thesis and recommend its acceptance: _________________________________ Erach R. Talaty, Committee Member _________________________________ Kandatege Wimalasena, Committee Member _________________________________ Lop-Hing, Ho, Committee Member iii ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Groutas for his kindness, guidance and support throughout my studies and research at Wichita State University, and for the opportunity given to me to broaden my knowledge and interest in organic chemistry and medicinal chemistry. I am very grateful for such a wonderful teacher in my life. I would also like to thank Dr. Talaty, Dr. Wimalasena and Dr. Ho for serving on my thesis committee. Furthermore, I would like to thank Dr. Alliston for his help with the biochemical studies. I am also thankful to members of my group and all the faculty and staff of the Department of Chemistry at Wichita State University. Many thanks must be extended to my family and friends for their love and support. iv ABSTRACT Chronic obstructive pulmonary disease (COPD) is a major health problem that affects 16 million people in the US, and is currently the fourth most common cause of death. Although the pathogenesis of COPD is poorly understood, current studies indicate that COPD is a multi-factorial disorder characterized by a cigarette smoke-induced cycle of oxidative stress, alveolar septal cell apoptosis, a protease/antiprotease imbalance, and chronic inflammation. An array of serine (HNE, PR3), cysteine (cathepsin S), and metallo- (MMP-1, MMP-9, MMP-12) proteases released by neutrophils, macrophages, and T lymphocytes contribute to the degradation of lung connective tissue and mediate a multitude of signaling pathways associated with the pathophysiology of the disorder. Re-establishment of a protease/antiprotease balance by utilizing potent and selective protease inhibitors is a promising approach for the development of potential therapeutics for COPD. We describe herein the design, synthesis and biochemical evaluation of a novel class of mechanism-based inhibitors of HNE that exploit the catalytic machinery of the target enzyme to generate a Michael acceptor. Subsequent reaction with an active site nucleophilic residue leads to inactivation of the enzyme. A noteworthy feature of the inhibitors is their ability to interact with the S 1-Sn’ subsites of the target enzyme. v TABLE CONTENTS Chapter Page 1. INTRODUCTION .......................................................................................................... 1 1.1 An Overview of COPD............................................................................................. 1 1.1.1 COPD................................................................................................................. 1 1.1.2 The Cause of COPD........................................................................................... 1 1.1.3 The Symptoms of COPD ................................................................................... 2 1.1.4. The Pathophysiology of Emphysema ............................................................... 2 1.2 An Overview of COPD-Relevant Proteases ............................................................. 3 1.2.1 Proteases ............................................................................................................ 3 1.2.1.1 Serine Proteases .......................................................................................... 3 1.2.1.2 Matrix Metalloproteases ............................................................................. 5 1.2.1.3 Cysteine proteases....................................................................................... 5 1.2.2 Elastin ................................................................................................................ 5 1.2.3 Antiproteinases .................................................................................................. 6 1.2.4 The Imbalance of Proteases and Antiproteases ................................................. 6 1.3 Inhibition................................................................................................................... 7 1.3.1 Substrate Specificity .......................................................................................... 7 1.3.2 Mechanism of Action....................................................................................... 10 1.3.3 Inhibition.......................................................................................................... 11 1.3.3.1 Irreversible inhibitors................................................................................ 11 1.3.3.2 Reversible inhibitors ................................................................................. 14 2. DESIGN RATIONALE AND RESEARCH GOALS .................................................. 17 2.1 Inhibitor Design Rationale...................................................................................... 17 2.2 Research Goals........................................................................................................ 21 3. EXPERIMENTAL........................................................................................................ 22 3.1 General.................................................................................................................... 22 3.2 Synthetic procedures............................................................................................... 22 3.3 Biochemical Assays................................................................................................ 35 4. RESULTS AND DISSCUSION................................................................................... 37 4.1 Synthesis ................................................................................................................. 37 vi TABLE CONTENTS (continued) Chapter Page 4.2 Biochemical Results................................................................................................ 39 5. CONCLUSIONS........................................................................................................... 41 REFERENCES ................................................................................................................. 42 vii LIST OF TALBES Table Page Table 1 Substrate Specifity of Enzymes Used in the Proposed Research.......................... 8 Table 2 Physical and Spectra Data of Compounds 1-9.................................................... 32 Table 2 Physical and Spectra Data of Compounds 1-9 (continued) ................................ 33 Table 2 Physical and Spectra Data of Compounds 1-9 (continued) ................................ 34 Table 3 Biochemical Evaluation of Inhibitors (I) ………………………………………39 viii LIST OF FIGURES Figure Page Figure 1 Berger and Schechter Nomenclature for Proteases ............................................. 7 Figure 2 The Serine Protease Reaction Mechanism ........................................................ 11 Figure 3 Kinetic Scheme for Mechanism-Based Inhibitors............................................. 12 Figure 4 Competitive Inhibition....................................................................................... 14 Figure 5 Kinetic Scheme of Reversible Inhibitors........................................................... 15 Figure 6 Human Neutrophil Elastase/Turkey Ovomucoid Inhibitor Complex................ 17 Figure 7 Network of Hydrogen Bonds and Hydrophobic Binding Interactions.............. 18 Figure 8 Structure-Based Design of 1, 2, 5-thiadiazolidin-3-one 1, 1-dioxide Scaffold . 19 Figure 9 3-Aza Grob Fragmentation Reaction................................................................. 20 Figure 10 Proposed Mechanism of Inhibition.................................................................. 21 Figure 11 Structures of Compounds 1-9.......................................................................... 28 Figure 11 Structures of Compounds 1-9 (continued)....................................................... 29 Figure 11 Structures of Compounds 1-9 (continued)....................................................... 30 Figure 11 Structures of Compounds 1-9 (continued)....................................................... 31 Figure 12 Time-Dependent Loss of Enzymatic Activity................................................. 40 Figure 13 Progress Curves of Inhibitor 8b with HNE ..................................................... 40 ix LIST OF SCHEMES Scheme Page Scheme 1 Synthesis of Inhibitors (I) ................................................................................ 38 x LIST OF ABBREVIATIONS AND TERMS COPD Chronic Obstructive Pulmonary Diseases NMR Nuclear Magnetic Resonance TLC Thin Layer Chromatography ClSO 2NCO Chlorosulfonyl isocyanate t-BuOH tertiary-Butyl alcohol TEA Triethylamine DCM Dichloromethane
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